We previously performed (in collaboration with NCATS, the NIH National Center for Advancing Translational Sciences) a genome-wide high-throughput siRNA screen of 21,500 cellular genes to identify ones involved in RSV infection. Human airway epithelial A549 cells in vitro were engineered to express Ds-Red as a viability marker, and were infected with RSV expressing enhanced green fluorescent protein (GFP) as a marker for viral gene expression. Three siRNAs per cellular gene were evaluated individually for reduction in GFP expression as a measure of inhibition of RSV infection. Computer analysis identified and corrected siRNA off-target effects. The top 155 hits (i.e., the greatest reductions in GFP without excessive loss of viability) were re-evaluated with at least three additional siRNAs each, and the top 65 hits were identified and confirmed manually. Among the genes identified for further analysis were four that encoded proteins associated with integrin signaling: actin-related protein 2 (ARP2); ADP-ribosylation factor 1 (ARF1); integrin alpha-V (ITGAV); and mitogen-activated protein kinase 3 (MAPK3). We previously reported results with ARP2, which was found to facilitate production and spread of progeny RSV particles to neighboring cells. In particular, ARP2 greatly increased the formation of filopodia, which are long slender projections from the cell surface that appeared to help deliver progeny RSV to neighboring cells. In other studies still in progress, siRNA-mediated knock-down of ARF1, ITGAV, and MAPK3 also reduced the production of progeny RSV. Two of the proteins, namely ARF1 and ITGAV, appeared to reduce the efficiency of RSV entry. Further studies of the underlying mechanisms are currently under way. Another of the top hits from the siRNA screen was the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase ion pump of the plasma membrane. Knock-down of ATP1A1 did not inhibit vesicular stomatitis virus, indicating that the inhibition was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also activated signaling from ATP1A1, which activated c-Src-kinase and transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block ATP1A1 signaling. PST2238 is a drug that already has been evaluated in clinical trials as a therapeutic to lower hypertension based on its effect on ATPIA1 signaling, and was shown to be well-tolerated in humans. Therefore, its a promising candidate for further evaluation as an antiviral therapeutic for RSV. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells and identified PST2238 as a potential anti-RSV drug. We previously performed codon-pair deoptimization (CPD) of various open reading frames (ORFs) of RSV. This is done by rearranging codons using computer algorithms and de novo gene synthesis to increase the content of normally-underrepresented codon-pairs without changing amino acid coding or overall codon usage. CPD is still a new technique and is incompletely understood. It typically has the effect of attenuation. This is thought to be due primarily to reduced efficiency of translation, although this is controversial and other mechanisms may also contribute. In previous years, we produced four RSV mutants by CPD of various combinations of ORFs, and showed that these viruses indeed were attenuated and, unexpectedly, were temperature-sensitive. We also evaluated the genetic stability of two of these viruses under restrictive temperatures, identified potential de-attenuating mutations, and used this information to make a promising vaccine candidate bearing a CPD L polymerase ORF that had desirable feature of attenuation, immunogenicity, and genetic stability. CPD RSV strains have been licensed for development to Codagenix, Inc. In the present report period, we characterized a previously-constructed CPD RSV mutant, Min B, in which the G and F surface glycoprotein ORFs had been subjected to CPD involving 619 nucleotide substitutions. This yielded a temperature sensitive virus that was highly restricted for replication in vitro. When subjected to selective pressure by serial passage at progressively increasing temperatures, Min B exhibited a partial restoration of replication fitness, expression of RSV F protein, and formation of syncytia. Comprehensive sequence analysis by whole-genome deep sequencing, coupled with long-range PCR, revealed the unexpected, abundant presence of viral genomes (named LD genomes) that contained large internal deletions and accumulated quickly during passage. The LD viral genomes ranged from 4.8-10.1 kb (32-67% of the RSV genome) and contained the CPD F gene in the first or second gene position rather than the usual eighth position. Thus, the deletions moved the CPD F gene to a promoter-proximal position that would increase the expression of F and thereby compensate for the CPD. Two representative LD viral genomes were chemically synthetized de novo. Phenotypic analysis revealed that both expressed high levels of F protein and complemented Min B replication in trans. Thus, LD viral genomes are internal-deletion defective genomes that enhanced rather than interfered with virus replication. These findings provide insight into the process of intracellular synthesis of RSV genomes. Specifically, these results suggest that the production of internal-deletion genomes is a very frequent event, but that these only become evident when they provide a selective advantage and are expanded. Thus, polymerase-jumping by the RSV polymerase probably is very frequent. Fortunately, this does not appear to pose any problem for RSV vaccinology, because LD genomes have not been sufficiently abundant to be detected in natural infection and, in any event, they do not provide any evident mechanism for increased pathogenesis. While this particular study with CPD virus did not provide further vaccine candidates, we are continuing to evaluate additional CPD viruses and have promising preliminary results that will be presented next year.